Selective protein degradation platforms have afforded new development opportunities for therapeutics and tools for biological inquiry. The first lysosome targeting chimeras (LYTACs) targeted extracellular and membrane proteins for degradation by bridging a target protein to the cation-independent mannose-6-phosphate receptor (CI-M6PR). Here, we developed LYTACs that engage the asialoglycoprotein receptor (ASGPR), a liver-specific lysosomal targeting receptor, to degrade extracellular proteins in a cell type-specific manner. We conjugated binders to a tri-GalNAc motif that engages ASGPR to drive downregulation of proteins. Degradation of EGFR by GalNAc-LYTAC attenuated EGFR signaling compared to inhibition with an antibody. Furthermore, we demonstrated that a LYTAC comprising a 3.4 kDa peptide binder linked to a tri GalNAc ligand degrades integrins and reduces cancer cell proliferation. Degradation with a single tri-GalNAc ligand prompted site-specific conjugation on antibody scaffolds, which improved the pharmacokinetic profile of GalNAc-LYTACs in vivo. GalNAc-LYTACs thus represent an avenue for cell-type restricted protein degradation.Users may view, print, copy, and download text and data-mine the content in such documents, for the purposes of academic research, subject always to the full Conditions of use:
A method has been developed for one-step ortho-selective ligand-directed H-D exchange, accompanied in some cases by concurrent acid-catalyzed electrophilic deuteration. This method is effective for deuteration of aromatic substrates ranging from ketones to amides and amino acids, including compounds of biological and pharmaceutical interest such as acetaminophen and edaravone. Use of a palladium catalyst featuring an NHC ligand is critical for the observed reactivity. Experimental evidence strongly suggests that palladium facilitates C-H activation of the aromatic substrates, a mechanism seldom observed under strongly acidic conditions. 2015 Elsevier Ltd. All rights reserved.
Fungal nonribosomal peptide synthetases (NRPSs) are megasynthetases that produce cyclic and acyclic peptides. In Aspergillus nidulans, the NRPS ivoA (AN10576) has been associated with the biosynthesis of grey-brown conidiophore pigments. Another gene, ivoB (AN0231), has been demonstrated to be an N-acetyl-6-hydroxytryptophan oxidase that putatively acts downstream of IvoA. A third gene, ivoC, has also been predicted to be involved in pigment biosynthesis based on publicly available genomic and transcriptomic information. In this paper, we report the replacement of the promoters of the ivoA, ivoB, and ivoC genes with the inducible promoter alcA in a single cotransformation. Co-overexpression of the three genes resulted in the production of a dark-brown pigment in hyphae. In addition, overexpression of each of the Ivo genes, ivoA-C, individually or in combination, allowed us to isolate intermediates and confirm the function of each gene. IvoA was found to be the first known NRPS to carry out the acetylation of the amino acid, tryptophan.
There is an urgent
need for point-of-care
tuberculosis (TB) diagnostic
methods that are fast, inexpensive, and operationally simple. Here,
we report on a bright solvatochromic dye trehalose conjugate that
specifically detects
Mycobacterium tuberculosis
(Mtb)
in minutes. 3-Hydroxychromone (3HC) dyes, known for having high fluorescence
quantum yields, exhibit shifts in fluorescence intensity in response
to changes in environmental polarity. We synthesized two analogs of
3HC-trehalose conjugates (3HC-2-Tre and 3HC-3-Tre) and determined
that 3HC-3-Tre has exceptionally favorable properties for Mtb detection.
3HC-3-Tre-labeled mycobacterial cells displayed a 10-fold increase
in fluorescence intensity compared to our previous reports on the
dye 4,4-
N,N
-dimethylaminonapthalimide (DMN-Tre).
Excitingly, we detected fluorescent Mtb cells within 10 min of probe
treatment. Thus, 3HC-3-Tre permits rapid visualization of mycobacteria
that ultimately could empower improved Mtb detection at the point-of-care
in low-resource settings.
Fungal
natural products (NPs) comprise a vast number of bioactive
molecules with diverse activities, and among them are many important
drugs. However, the yields of fungal NPs from native producers are
usually low, and total synthesis of structurally complex NPs is challenging.
As such, downstream derivatization and optimization of lead fungal
NPs can be impeded by the high cost of obtaining sufficient starting
material. In recent years, reconstitution of NP biosynthetic pathways
in heterologous hosts has become an attractive alternative approach
to produce complex NPs. Here, we present an efficient, cloning-free
strategy for the cluster refactoring and total biosynthesis of fungal
NPs in Aspergillus nidulans. Our platform places
our genes of interest (GOIs) under the regulation of the robust asperfuranone afo biosynthesis gene machinery, allowing for their concerted
activation upon induction. We demonstrated the utility of our system
by creating strains that can synthesize high-value NPs, citreoviridin
(1), mutilin (2), and pleuromutilin (3), with good to high yield and purity. This platform can
be used not only for producing NPs of interests (i.e., total biosynthesis)
but also for elucidating cryptic biosynthesis pathways.
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